Samples of blood are often collected for use in various applications such as diagnostics. Traditionally, the blood was collected in order to look at cellular morphology and to count cells. Conventional blood collection involves collecting the blood into tubes which contain various additives such as EDTA, heparin or citrate. These additives function as anticoagulants which allow for the blood samples to be stored for longer periods of time prior to analysis than if collected without the use of any additive. Currently, diagnostics is shifting towards a focus on molecular diagnostics, in which nucleic acids are isolated and studied. The analysis may include the use of various different methods including polymerase chain reaction (PCR), reverse transcription PCR (RT PCR), real time PCR, RNA and DNA chips, gene expression arrays, microarrays, and restriction fragment length polymorphism (RFLP) among others.
Blood is still a key biological sample which is being used for diagnostics, however, the traditional additives used for the preservation of blood offer a number of disadvantages when nucleic acids are to be isolated and analyzed. Nucleic acids in blood, and particularly RNA, are very unstable. The key to the successful analysis of nucleic acids isolated from blood is that the nucleic acid is of a high quality and that it remains intact from the time of sample collection until the nucleic acids can be isolated and analyzed in the lab. Once a blood sample has been obtained from an individual, a major problem is the stability of the nucleic acids within the sample. Nucleic acids in a biological sample quickly degrade at ambient temperature. Cells contain nucleases which quickly degrade and destroy nucleic acids as soon as they come into contact with DNA or RNA substrates. These nucleases are controlled due to their location in various compartments within the cells including lysosomes. However, when blood samples are drawn, the cells present in the blood will start to die, and these nucleases will be released from the lysosomes and will quickly begin to degrade the nucleic acids within the blood sample. Thus, this is a major problem with using blood samples as a source of nucleic acids for analysis. The stability of the nucleic acids within the blood sample must be maintained as this determines whether the nucleic acids can be successfully analyzed, be it for research or diagnostic purposes. Traditional blood preservatives, such as EDTA and heparin, do not allow for the preservation of the nucleic acids present within the blood sample and thus the DNA, and more specifically RNA, will quickly degrade in the presence of these additives.
In recent years, the study of gene expression has also been increasing, with gene activity and nucleic acids obtained from biological samples being used to diagnose infections or diseases including cancer, and to monitor the effects of administered drugs, among other applications. Therefore, in addition to the isolation of high quality nucleic acids from blood, it is also of interest to inhibit or block gene induction in an isolated blood sample such that the “snapshot” of the gene expression and levels at the moment the blood sample is drawn is maintained. When a sample of blood is drawn induction of gene transcription may occur, with over-production or under-production of some mRNA species. This will result in changes in the transcript pattern of the sample and subsequent analysis of gene expression will therefore be altered. Therefore inhibiting or blocking gene induction immediately upon the collection of blood samples is highly important for many downstream applications of the blood sample. Again, traditional blood preservation additives such as EDTA and heparin will not function to inhibit gene induction.
Another important aspect of preserving blood samples for use in molecular applications is that the sample should be preserved at room temperature for extended periods of time in order to allow for ease of shipping of the sample. Samples are often collected in one location, and then shipped to another location for analysis. If a blood sample can be stored and shipped at room temperature while preserving the integrity of the nucleic acids this would be of a great benefit in terms of ease of shipping and shipping costs. As well, this would be a key for blood samples which are collected in remote locations or in resource-limited settings where refrigerators and freezers for storage of the blood samples may not be available.
Another major concern with the collection and transport of blood samples for nucleic acid analysis is that these samples are often infectious, as the sample may contain live virus or bacteria. The presence of live infectious pathogens in these biological samples poses a health and safety risk to the individuals involved in the collection, transfer and testing of the samples if the samples are kept viable and/or biologically intact. Due to the potential dangers of shipping biologically intact samples the expense and effort required in shipping these samples is increased.